Scotti Nunzia, Cardi Teodoro
Res. Division Portici, CNR-IGV, National Research Council of Italy, Institute of Plant Genetics, Portici, NA, Italy.
Methods Mol Biol. 2012;847:451-66. doi: 10.1007/978-1-61779-558-9_35.
The production of biopharmaceuticals in plants is currently one of the most attractive approaches to modern medicine. Several efficient plant-based expression systems have been developed so far. Among them, plastid transformation has attracted biotechnologists because the plastid genome, unlike nuclear genome, bears a number of unique advantages for plant genetic engineering. These include higher levels of protein production, uniform gene expression of transformants due to the lack of epigenetic interference, and expression of multiple genes (as in operons) from the same construct. Further, the plastid transformation technology is an environmentally friendly method because plastid and their genetic information are maternally inherited in many species with a consequent lack of transmission of plastid DNA by pollen. Recently, great progress has been made with plastid-based production of biopharmaceuticals demonstrating that it is a promising platform for such purposes. This chapter describes detailed protocols for plastid transformation including the delivery of DNA by biolistic method, the selection/regeneration of transplastomic plants, and the molecular analyses to select homoplasmic plants and confirm transgene expression.
利用植物生产生物制药目前是现代医学中最具吸引力的方法之一。到目前为止，已经开发出了几种高效的基于植物的表达系统。其中，质体转化吸引了生物技术学家，因为与核基因组不同，质体基因组在植物基因工程方面具有许多独特优势。这些优势包括更高水平的蛋白质生产、由于缺乏表观遗传干扰而使转化体的基因表达均匀，以及从同一构建体中表达多个基因（如操纵子中的基因）。此外，质体转化技术是一种环境友好型方法，因为在许多物种中质体及其遗传信息是母系遗传的，因此花粉不会传播质体DNA。最近，基于质体生产生物制药取得了巨大进展，表明它是实现此类目的的一个有前景的平台。本章描述了质体转化的详细方案，包括通过基因枪方法导入DNA、转质体植物的选择/再生，以及用于选择同质性植物和确认转基因表达的分子分析。
